Optical biosensor referencing method

ABSTRACT

A referencing method for an optical biosensor system using a single sensing region is provided. The method involves limiting the ligand immobilized in a single sensing region to only a portion thereof. In one embodiment, this is accomplished by selectively deactivating a portion of the sensing surface to prevent immobilization of ligand to that portion. As a result, a reference response can be recorded in the same sensing region as a molecular interaction response. Thus, the bulk refractive index can be accurately accounted for in measuring the kinetics of a molecular interaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/589,731 filed on Jan. 23, 2012.

BACKGROUND

Surface plasmon resonance (SPR) based biosensors are commonly used toperform kinetic studies of complex molecular interactions such asbetween hormone-receptor, enzyme-substrate and antigen-antibody. Thebiosensors are typically in the form of one or more sensing regionshoused within a flow cell of a microfluidic system. The microfluidicsystem defines a series of flow paths that direct fluid flow to the flowcell containing the sensing regions. The one or more sensing regions ofthe flow cell support immobilized molecules referred to as “ligands.”The ligands bind molecules known as “analytes” which are present influids that are directed to the sensing region via the microfluidicsystem. Current analysis methods determine the kinetic interaction ofthe ligand and analyte by separately injecting a series of analyteconcentrations into the system and measuring the change in refractiveindex at the sensing regions. Based on the changes in refractive index,one can determine the real-time kinetics of the interaction between theanalyte and ligand, including association and dissociation rates.

When the analyte being tested consists of a small molecule, the changein refractive index at the sensing region due to interaction with ligandis typically very small and may be difficult to detect above therefractive index from the bulk flow of the sample in the sensing region(bulk refractive index). This is typically a result of highconcentrations of the small molecule or more commonly, highconcentrations of solubilizing agent such as dimethylsulfoxide (DMSO).As such, a referencing method is needed that allows the response fromthe bulk refractive index to be separated from the response due to themolecular interaction between analyte and immobilized ligand at thesensing region.

The double referencing method has been one approach to solving thisproblem. In this method, the sample is caused to flow over two separatesensing regions; one of the sensing region contains immobilized ligand(working sensing region) and one sensing region is free of immobilizedligand (reference sensing region). Thus, the response from the referencesensing region can be subtracted from the response in the workingsensing region to yield the response attributed to the molecularinteraction in the working sensing region.

However, there are some limitations associated with this particularmethod. Differences, such as temperature, sensitivity and dispersion,between the working sensing region and reference sensing region cansignificantly impact the data quality and obscure the binding response,especially with low molecular weight molecules. Dispersion of the samplethat may occur between the two sensing regions is of a particularconcern. In most microfluidic systems, a buffer fluid will typicallyflow through the channels housing the sensing regions prior to exposureto the fluid containing the analyte sample. Since the sensing regionsare separated, the sample is likely to encounter additional residualbuffer in the flow path from the reference sensing region to the workingsensing region. This causes dilution of the sample thereby changing therefractive index such that the response obtained from the referencesensing region is not entirely an accurate representation of the samplethat encounters the working sensing region. These concerns areparticularly pertinent when the bulk refractive index of the sample andbuffer differ, even where the difference is small (e.g. 500 RU). Thesesmall differences can have profound effects on the accuracy of thekinetic calculations for the association and dissociation rates of thebinding interactions between analyte and ligand. Thus, a method isneeded that provides a reference measurement without the deleteriouseffects of a two sensing region referencing system.

SUMMARY

A method is provided that allows a single sensing region to be dividedinto two or more sub-regions, wherein one sub-region provides areference measurement and one sub-region provides an interactionmeasurement thereby alleviating many of the problems associated with aseparate reference sensing region. The method comprises the steps ofactivating an entire sensing region to permit immobilization of a ligandthereon, selectively deactivating a portion of the sensing region toprevent immobilization of a ligand as to the sub-region, and injectingthe ligand over the sensing region to permit immobilization onto theportion of the activated portion of the sensing region. Thus, the methodprovides a single sensing region that has been divided into twoportions, wherein a first portion provides a reference measurement (theportion that has been deactivated) and a second portion provides aninteraction measurement (the activated portion containing the ligand).Specifically, the first portion encompasses an area of the sensingregion that is interrogated by low surface plasmon resonance angles andthe second portion encompasses an area of the sensing region that isinterrogated by high surface plasmon resonance angles. Since thereference portion is in the same sensing region with the workingportion, the concerns with dispersion and differences between separatesensing regions are eliminated, or at the least, minimized, therebyproviding a more accurate referencing method. Additionally, the presentmethod increases the data capability of a biosensor system since eachsensing region in a microfluidic flow cell can be modified to includeits own integrated reference point alleviating the need to use an entireseparate sensing region for a reference point thereby increasing assaycapacity with minimal added complexity.

In one embodiment, a method for providing a reference bulk refractiveindex response and a binding response in a single sensing regiondisposed along a channel in a flow cell of a biosensor system isprovided. In the present embodiment, a first sample is injected througha first input port at a first end of a sensing region. The first samplecomprises an activating agent sufficient to permit immobilization of aligand onto the surface of the sensing region. The first sample flowsfrom the first end of the sensing region to a second region where itexits via a second exit port. Once the surface of the sensing region hasbeen sufficiently exposed to the sensing region, the injection of thefirst sample is terminated. A second sample is then injected at a flowrate through the first input port, wherein a first exit port located onthe first end of the sensing region is opened and the second exit portis closed. The flow rate of the second sample is then modified in amanner sufficient to cause the second sample to contact only a firstportion of the sensing region thereby defining a second portion of thesensing region that is not contacted by the second sample, wherein thesecond sample comprises a deactivating agent sufficient to render thefirst portion incapable of having ligand immobilized thereon. The extentof the migration of the second sample along the sensing region isselected to position the first portion in an area of the sensing regionthat is interrogated by low surface plasmon resonance angles such thatthe second portion encompasses an area of the sensing region that isinterrogated by high surface plasmon resonance angles. Followingtermination of the second sample injection, a third sample comprisingthe ligand is injected through the first input port with the second exitport open and the first exit port closed such that first and secondportions of the sensing region are exposed to the third sample. However,due to deactivation of the first portion, the ligand is only immobilizedto the second portion of the sensing region. The third sample injectionis terminated and thereafter, the analyte sample is injected through thefirst input port and caused to flow over the first and second portionsof the sensing region. The responses at the first and second portions ofthe sensing region are recorded wherein the response at the firstportion provides a reference response comprising the bulk refractiveindex and the response at the second portion includes the bindingresponse resulting from the interaction between analyte and ligand. Thereference response is then subtracted from response at the secondportion to isolate the response elicited from the binding interaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts and injection profile of three independent sensingregions represented by curves 1, 2, and 3. Curves 1 and 3 representinjection profiles at sensing regions that were independently addressedwhereas curve 2 represents an injection profile at a sensing regionfollowing exposure to a separate sensing region.

FIG. 2 depicts a set of exemplary responses elicited by a serialdilution. The curves in the right panel represent the bulk refractiveindex recorded at a reference sensing region and demonstrate thedispersion effect. The curves in the left panel represent the analytebinding curves at a separate working sensing region.

FIG. 3 provides an exemplary kinetic model fit for the bindinginteractions of the left panel of FIG. 2 with the reference responsefrom the right panel of FIG. 2 subtracted.

FIG. 4 depicts an exemplary low mass SPR dip mapped to a sub-region of asensing region in a flow cell.

FIG. 5 depicts an exemplary high mass SPR dip mapped to a sub-region ofa sensing region in a flow cell.

FIG. 6 depicts and exemplary high mass SPR dip and low mass SPR dipmapped to different sub-regions of a sensing region in accordance withthe methods of the present invention.

FIG. 7 depicts a flow cell with a serpentine channel having threeseparate sensing regions disposed thereon.

FIG. 8 depicts a sub-addressing method used with the methods of thepresent invention.

FIG. 9 provides an illustration of one embodiment of the inventivemethod.

DETAILED DESCRIPTION

The ability to measure binding of small drug molecules to immobilizedproteins in real time has become feasible mainly as a result of improvedsignal referencing methods such as the double referencing method.However, this method has some limitations related to how similar thereference sensing region is with respect to the working sensing region.Differences in temperature, sensitivity and dispersion at each locationcan have a significant impact on the data quality and can obscure thebinding response expected from low molecular weight molecules. Theseconcerns are extremely pertinent when the bulk refractive index of thesample and the continuous flow buffer differ by even a small amount(e.g. 500 RU). A particularly difficult issue is dispersion. This iswhere the dead volume that exists between the working sensing region andthe referencing sensing region is sufficient to cause dilution of thesample and hence cause changes in the bulk refractive index injectionprofile that then translates into distortions of the double referenceddata.

FIG. 1 shows a zoom view of an injection profile focusing on the plateauthat forms during the injection (i.e. shaded region). As depictedtherein, the second sensing region (curve 2) possesses far morecurvature than the other two sensing regions. This curvature onlyrepresents 2% of the total bulk refractive index response, but has aserious negative impact on data quality when performing a kineticanalysis. The curvature in the second sensing region is caused by mixingwith the running buffer that is present at an exit port located betweenthe sensing regions.

The effect of this curvature in the injection profile appears in thedouble referenced response as an apparent binding signal as shown inFIG. 2. The curves in the right panel show the dispersion effectisolated for a series of dilutions. The curves in the left panelrepresent the analyte binding curves that are distorted by thedispersion. If the dispersion effect is subtracted, the data set qualityis improved to enable a reasonable kinetic model fit as shown in FIG. 3.

As indicated by FIGS. 1-3, the contribution of dispersion to the rawresponse should be accounted for to obtain accurate results. However,the double referencing method is imperfect and the residual error isincreased if the refractive index difference between the buffer and thesample is increased. The accuracy of the subtraction to correct theresponse depends on matching the working and reference sensing spots asclosely as possible in terms of temperature, position, sensitivity, massloading etc. In current SPR systems based on SPR imaging, it is possibleto obtain response readings from any region of the plane that is beingprobed by the SPR such that accurate referencing should be moreconvenient. However, SPR imaging does not possess the sensitivity ofmonochromatic angle-based SPR interrogation where an elongated stripregion within the flow cell is interrogated. Generally, flow cells ofbiosensor systems include several separate planes or sensing regionsthat are being actively probed by the SPR. As used herein, the term“sensing region” refers to a single region in a flow cell that is beingactively probed by the surface plasmon resonance over a single range ofincreasing reflectance angles.

When nothing has been immobilized onto a surface of a sensing region,the photodiode array returns a single surface plasmon resonance dip. Thepixels correlate to the SPR angles that match the resonance state whenthere is no additional mass loaded onto the SPR sensing surface. Theresonance maps onto a particular area of sensing region 8 of flow cell10 as in FIG. 4. It is usual to design the SPR detector to allow an SPRresonance to be measured between a refractive index of 1.333 to 1.40.When mass loading is low, the average refractive index at the sensingregion will be very close to 1.334 and the SPR resonance dip will appearto one side of the photodiode array detector.

When a large amount of mass has been loaded onto the surface, thephotodiode array returns a single surface plasmon resonance dip shiftedto different SPR angles that map onto a region of the flow cell that isfar from the original position before mass was loaded. The SPR dip hasshifted to the right due to possessing a higher average refractive indexresulting from mass loading as shown in FIG. 5.

When interrogating the SPR signal only the bottom 30% of the dip isrequired for computations and so the remainder of the SPR scan is notrequired. This type of monochromatic angle-based SPR detector is commonand a single SPR dip is found per sensing region where each sensingregion covers an angular range that corresponds to a refractive indexmeasuring range of 1.333 to 1.40.

Thus, if the SPR resonance obtained when no mass is loaded on thesensing region is maintained in the presence of the second SPR resonancefor high mass loading, then the low mass resonance provides a suitablereference that is very closely matched to the high mass loadingresonance. This is demonstrated in FIG. 6.

A common approach is to include adjacent, but separate sensing regionsthat are passed in series or in parallel by the liquid stream, but eachsensing region would itself also cover the entire measuring range from1.333 to 1.40. Under these circumstances, a minimum physical separationdistance is unavoidable. This distance can be reduced if the sensingregions are in parallel and housed in the same flow cell. However, thiswould require hydrodynamic addressing of each sensing spot within thesame flow and can be complicated by cross-contamination issues. Oneapproach to address these issues is through the use of sequentialsensing regions connected in a series using a serpentine channelconfiguration such as flow cell 11 depicted in FIG. 7.

As shown in FIG. 7, flow cell 11 consists of a serpentine channel 12possessing three sensing regions 14. Input/output ports 16 arepositioned on each side of sensing regions 14 with separate input ports18 and output ports 20 positioned at each end of serpentine channel 12.Shaded region 22 provides the mapped location of the SPR dip. Sensingregions 14 can have a length of about 3 mm, but can be much shorter, orlonger, depending on the detector design. It is important to note thatthe placement of ports 16, 18, 20 which allow independent fluidaddressing to each sensing region 14, but they do not allow localizedaccess to sub-regions within sensing regions 14.

Localized fluidic access to sub-regions of a given sensing region 14would permit the low mass SPR dip to be attained in the presence of thehigher mass SPR dip. This enables dispersion effects to be subtractedwhen a large bulk refractive index variation between samples and bufferexists, which is common in drug discovery where high concentrations ofDMSO are needed for solubility and standard assay plate preparation doesnot allow for bulk refractive index matching. The ability to accuratelyreference out the bulk refractive index effects allows resolution of lowbinding responses even when large bulk refractive index mismatches existbetween sample and running buffer.

Localized fluidic addressing can be accomplished by adding moreinput/output ports 16 along each sensing region 14. However, thisapproach complicates the fluidic control systems required and moreimportantly, dispersion at these new ports can interfere with idealreferencing between the two resonance dips in a single sensing region.The present methods provide a more effective solution by creating a lowmass and high mass SPR dip in a single sensing region to avoid thedispersion effects.

In conventional approaches, sensing regions either display a low massSPR dip due to the complete absence of immobilized ligand or a high massSPR dip due to immobilized ligand at high density. However, the presentmethod permits both a low mass SPR dip and high mass SPR dip byselectively confining the ligand to a sub-region of the sensing regionswhere the high mass dip would be expected, thereby permitting a low massdip in the sub-region void of ligand. In this instance, the low mass dipis very closely matched to the high mass dip as it is separated by avery small distance and is contained in the same sensing region.Moreover, this approach does not require the presence of fluid accessholes disposed between the dip regions thereby greatly reducingdispersion that might occur between these two sub-regions. Accordingly,the dispersion experienced by sample flowing over both SPR dip regionsin a single sensing region will be practically identical allowing veryaccurate double referencing and higher resolution of low molecularweight analyte binding.

In one embodiment, ligand is prevented from being loaded onto an entiresensing region and is confined to a sub-region where high mass loadingresonance (i.e., high surface plasmon resonance angles) is expected toexist thereby providing a dual resonance where one resonance can act asan almost perfect reference for the other. This can be accomplished bylocalized ligand immobilization within a sub-region of a given sensingregion by exploiting the behavior of a laminar flow stream as describedbelow and depicted in FIG. 8.

FIG. 8 provides a time progression of a sample flow path (shaded area)containing the desired ligand to be immobilized on a sub-region ofsensing region 40 in channel 30 in a single flow cell design. Channel 30includes first input port 32, second input port 34, first exit port 36,second exit port 38, and sensing region 40 provided between first andsecond input ports 32, 34. Sample containing the desired ligand (shadedarea) is injected into first input port 32 where channel 30 ispre-filled with a given buffer (not shown) before commencing theinjection. At the first time point T1, first exit port 36 is open thusforcing the sample to exit immediately adjacent to first input port 32.However, by adjusting the sample flow rate, the sample interface frontwill migrate down channel 30 towards second input port 34 as shown attime points T2, T3 and T4 thereby enabling precise control of theposition of the sample interface within the flow cell. By preciselycontrolling the flow rate, a counterflow stream is not necessary tocontrol the position of the interface. Thus, the interface will simplycontinue to migrate down channel 30 until such time as the sample flowis stopped at a pre-calculated target position. Channel 30 is thenpurged with sample free buffer entering from second input port 34 andexiting at first exit port 36. In a symmetrical system, it is possibleto execute this same positional control (also referred to herein as“sub-addressing”) from the opposite side or even for multiple sensingregions connected in series as in the serpentine flow cell shown in FIG.7. Additionally, a flow cell suitable for use with the present inventivemethod is described in U.S. Pat. No. 8,298,496, which is incorporatedherein by reference.

The sub-addressing procedure described above in connection with FIG. 8permits selective loading of ligand to a sub-region of the sensingregion thereby providing a dual resonance measurement in a singlesensing region as depicted in FIG. 6.

One embodiment of the current invention is depicted in FIG. 9. In thisembodiment, the first step of the present method involves injecting anactivating agent (light gray shading) through first input port 32 andcausing the activating agent to flow over the entire sensing region 40to second exit port 38. The second step involves injecting adeactivation solution (darker gray shading) through first input port 32and using the sub-addressing procedure to selectively deactivate asub-region of sensing region 40. In performing this step, thedeactivating agent should be prevented from migrating to the sub-regionof the sensing region that is expected to be interrogated by high SPRangles. The third step involves injecting the desired ligand (dottedarea) into the channel 30 via first input port 32 and exiting via secondexit port 38. As a result, the ligand injected will only be covalentlybound to the high mass loading region (i.e. area interrogated by highSPR angles) that remains activated as shown in step 4. In this case, anethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)mixture could be used to activate carboxyl groups available at thesensing surface and a deactivation reagent such as sodium hydroxide orethanolamine could be employed.

It is imperative that the amount of mass loaded onto the high massregion is sufficient to generate a resonance in that region. For examplea 20 pixel shift to the right of the photodiode array (represents about16,000 RU immobilized) would cause two clear SPR resonances to appearthat are well resolved and can function as described. If theimmobilization falls short of the target level then an additive (e.g.sucrose, glycerol etc) can be added to the buffer to shift the resonanceto the correct region by making up the refractive index deficit requiredto map the resonance dip to the coated region.

The present method can be exploited in a variety of different manners toyield a dual resonance sensing region. For example, the entire sensingregion 40 can be first exposed to an activating agent as described aboveand then the sub-addressing procedure can be used to restrict ligand toa particular sub-region. Alternatively, the sub-addressing procedure canbe used to expose a sub-region of the sensing region to an activatingagent thereby only permitting ligand loading as to the activatedsub-region.

In another embodiment, rather than forming multiple resonances bychanging the mass density at sub-regions in the sensing region, it isalso possible to expose each sub-region to separate flow wherein therefractive index is adjusted such that a resonance dip exists for eachstream intersecting the sensing region at right angles.

In another embodiment, ligand mass loading can be supplemented byco-immobilizing other non-interacting species (e.g. ovalbumin) that canconfer the required mass increase yet remain neutral andnon-interfering. For example, if the ligand is a low MW peptide theneven at maximum immobilization capacity it will not be possible toobtain sufficient immobilized mass. The peptide could first be preparedwith a biotinylated terminal group and then conjugated to a biotinbinding protein such as neutravidin and this high molecular weightcomplex can then be immobilized at sufficient density.

In yet another embodiment, the second low refractive index resonanceregion may in fact be coated with a low amount of non-specific ligand orspecific ligand and function as either a reference or an additionalworking sensing region. The sub-region fluidic addressing procedure canbe used to immobilize ligands that are confined to either high or lowmass regions. In this manner, a single conventional sensing region thatcovers the full measuring range can be subdivided into two or moresub-regions that can each maintain an SPR resonance and each can be usedas either reference or working sensing regions.

In yet another embodiment, the prism through which light undergoes totalinternal reflection in order to generate the SPR signal can be composedby segments of optical substrates (e.g. glass, plastic, sapphire,quartz) with different dielectric properties such that each adjacentdielectric substrate is arranged such that it will present a differentSPR resonance condition at the sensing surface allowing the SPR dips tobe shifted with respect to each other without changing the loaded massor the bulk refractive index at the sensing surface. In particular anelectro-optical material that can exhibit the required birefringencecould be employed. An on-demand mechanical mechanism where differentprism segments (or exchange entire prism) can be exchanged within theoptical detector could also be used.

Thus, the present invention is well adapted to carry out the objects andattain the ends and advantages mentioned above as well as those inherenttherein. While preferred embodiments of the invention have beendescribed for the purpose of this disclosure, changes in theconstruction and arrangement of parts and the performance of steps canbe made by those skilled in the art, which changes are encompassedwithin the spirit of this invention as defined by the appended claims.

What is claimed is:
 1. A method for providing a reference bulkrefractive index response and a binding response in a sensing regiondefined along a channel in a flow cell of a biosensor system comprisingthe steps of: injecting a first sample through a first input port at afirst end of the sensing region wherein a second exit port at a secondend of the sensing region is open and a first exit port at the first endof the sensing region is closed such that the first sample is caused toflow from the first end of the sensing region to the second end of thesensing region, wherein the first sample comprises an activating agentsufficient to permit immobilization of a ligand; terminating theinjection of the first sample; injecting a second sample at a flow ratethrough the first input port, wherein the first exit port is opened andthe second exit port is closed; modifying the flow rate of the secondsample in a manner sufficient to cause the second sample to contact afirst portion of the sensing region thereby defining a second portion ofthe sensing region that is not contacted by the second sample, whereinthe second sample comprises a deactivating agent sufficient to renderthe first portion unable to immobilize the ligand; terminating theinjection of the second sample; injecting a third sample comprising aligand through the first input port with the second exit port open andthe first exit port closed such that first and second portions of thesensing region are exposed to the third sample, wherein the ligand isonly immobilized to the second portion of the sensing region due todeactivation of the first portion; and terminating the injection of thethird sample.
 2. The method of claim 1 wherein the first portionencompasses an area of the sensing region that is interrogated by lowsurface plasmon resonance angles and the second portion encompasses anarea of the sensing region that is interrogated by high surface plasmonresonance angles.
 3. The method of claim 1 wherein the activating agentis a mixture of ethyl(dimethylaminopropyl) carbodiimide andN-hydroxysuccinimide.
 4. The method of claim 1 wherein the deactivatingagent is selected from the group consisting of sodium hydroxide andethanolamine.
 5. The method of claim 1 wherein the flow cell containsadditional sensing regions and the method is repeated for eachadditional sensing region.
 6. The method of claim 1 wherein the ligandis immobilized to the second portion at a density sufficient to generatea high mass surface plasmon resonance dip.
 7. The method of claim 1wherein the third sample further comprises a non-interacting agent thatco-immobilizes with the ligand at the second portion.
 8. The method ofclaim 7 wherein the ligand is of a low molecular weight.
 9. A method formeasuring a reference bulk refractive index response and a bindingresponse in a sensing region defined along a channel in a flow cell of abiosensor system comprising the steps of: injecting a first samplethrough a first input port at a first end of the sensing region whereina second exit port at a second end of the sensing region is open and afirst exit port at the first end of the sensing region is closed suchthat the first sample is caused to flow from the first end of thesensing region to the second end of the sensing region, wherein thefirst sample comprises an activating agent sufficient to permitimmobilization of a ligand; terminating the injection of the firstsample; injecting a second sample at a flow rate through the first inputport, wherein the first exit port is opened and the second exit port isclosed, and wherein the second sample comprises a deactivating agentsufficient to render the first portion unable to immobilize the ligand;modifying the flow rate of the second sample in a manner sufficient tocause the second sample to contact a first portion of the sensing regionthereby defining a second portion of the sensing region that is notcontacted by the second sample, and wherein the first portionencompasses an area of the sensing region that is interrogated by lowsurface plasmon resonance angles and the second portion encompasses anarea of the sensing region that is interrogated by high surface plasmonresonance angles; terminating the injection of the second sample;injecting a third sample comprising the ligand through the first inputport with the second exit port open and the first exit port closed suchthat first and second portions of the sensing region are exposed to thethird sample, wherein the ligand is only immobilized to the secondportion of the sensing region due to deactivation of the first portion;terminating the injection of the third sample. injecting a fourth samplethrough the first input port wherein the second exit port is open andthe first exit port is closed such that the fourth sample is caused toflow over the first and second portions of the sensing region, whereinthe fourth sample comprises an analyte; measuring a first response atthe first portion of the sensing region and a second response at thesecond portion of the sensing region as the fourth sample flows over thesensing region; and subtracting the first response from the secondresponse to determine a binding response attributed to the interactionof the analyte with the ligand.
 10. The method of claim 9 wherein theligand is immobilized to the second portion at a density sufficient togenerate a high mass surface plasmon resonance dip.
 11. The method ofclaim 9 wherein the fourth sample further comprises an additive in anamount sufficient to shift the resonance to elicit a high mass surfaceplasmon resonance dip at the second portion while maintaining a low masssurface plasmon resonance dip at the first portion.
 12. The method ofclaim 11 wherein the additive is selected from the group consisting ofsucrose and glycerol.
 13. The method of claim 9 wherein the third samplefurther comprises a non-interacting agent that co-immobilizes with theligand at the second portion.
 14. The method of claim 13 wherein theligand is of a low molecular weight and the non-interacting agent isovalbumin.
 15. A method for measuring a bulk refractive index and abiomolecular interaction in a single sensing region of a biosensingsystem comprising the steps of: injecting a sample comprising an analytethrough a flow cell comprising a sensing region, wherein a first portionof the sensing region has a ligand immobilized thereon and a secondportion of the sensing region is free of immobilized ligand, and whereinthe first portion encompasses an area of the sensing region that isinterrogated by high surface plasmon resonance angles and the secondportion encompasses an area of the sensing region that is interrogatedby low surface plasmon resonance angles; measuring a first response atthe first portion and a second response at the second portion as thesample flows over the sensing region; and subtracting the secondresponse from the first response to determine a binding responseattributed to the interaction of the analyte with the ligand.
 16. Themethod of claim 15 wherein the ligand is immobilized to the firstportion at a density sufficient to generate a high mass surface plasmonresonance dip.
 17. The method of claim 15 wherein the sample furthercomprises an additive in an amount sufficient to shift the resonance toelicit a high mass surface plasmon resonance dip at the first portionwhile maintaining a low mass surface plasmon resonance dip at the secondportion.
 18. The method of claim 17 wherein the additive is selectedfrom the group consisting of sucrose and glycerol.
 19. The method ofclaim 15 wherein the first portion includes a non-interacting agent thatis co-immobilized with the ligand.
 20. The method of claim 19 whereinthe ligand is of a low molecular weight and the non-interacting agent isovalbumin.